Methods for producing factor VIII proteins

ABSTRACT

Methods are provided for purification of Factor VIII polypeptides by immunoaffinity chromatography and ion exchange chromatography, in which the eluate from the immunoaffinity column is diluted with a solution comprising higher salt concentration, or lower non-polar agent concentration than that of the elution solution, prior to passing the diluted solution through the ion exchange column. The methods result in improved purification without significant yield loss.

FIELD OF THE INVENTION

[0001] The present invention relates to improved methods for thepurification of procoagulant proteins, particularly recombinantproduction of Factor VIII and related proteins.

BACKGROUND OF THE INVENTION

[0002] Hemophilia is an inherited disease which has been known forcenturies, but it is only within the last few decades that it has beenpossible to differentiate among the various forms; hemophilia A andhemophilia B. Hemophilia A is caused by strongly decreased level orabsence of biologically active coagulation factor VIII, which is aprotein normally present in plasma.

[0003] Until recently, therapeutic factor VIII concentrates have beenprepared by fractionation of plasma. However, in recent years, DNAsequences encoding the human coagulation cofactor, Factor VII:C (FVM),became known in the art [see e.g., Toole et al, 1984, Nature312:312-317; Wood et al, 1984, Nature 312:330-337; Vehar et al, 1984,Nature 312:337-342], as well as methods for expressing them to producerecombinant FVIIE [see e.g. Toole, U.S. Pat. No. 4,757,006; WO 87/04187,WO 88/08035 and WO 88/03558]. Active variants and analogs of FVIIprotein, and DNA sequences encoding them, have also been reported [seee.g. Toole, U.S. Pat. No. 4,868,112; EP 0 786 474; WO 86/06101 and WO87/07144]. Generally, such variants and analogs are modified such thatpart or all of the B domain are missing and/or specific amino acidpositions are modified, for example, such that normally protease-labilesites are resistant to proteolysis, e.g. by thrombin or activatedProtein C. Other analogs include modification at one or more lysineand/or tyrosine residues.

[0004] It has been previously surprisingly found that the B domain isdispensable for the procoagulant activity of FVIII, and that activeprocoagulant protein can be expressed and secreted by expression of aFVIII-encoding DNA in which the nucleotide region encoding part or allof the B domain is lacking. Not only is active protein of these variantsproduced and secreted, it accumulates in the media at higher levels thanwhen expressed by the full-length DNA. The reduced level of activeprocoagulant FVVI protein in the media has been attributed, at least inpart to several factors [see e.g. WO 87/04187, WO 88/08035 and WO88/03558]. In Toole et al., Exploration of Structure-FunctionRelationships in Human Factor VIII by Site-directed Mutagenesis, ColdSpring Harbor Symposium on Quantitative Biology, 51:543 (1986), it wasreported that recombinant FVIII could be purified by a combination ofmonoclonal antibody affinity chromatography and ion-exchangechromatography. U.S. Pat. No. 5,470,954 describes a similar process inwhich FVIH is passed directly from immunoaffinity purification to theion exchange column. In that document it is stated that changing theionic strength of the eluted polypeptide solution increases the chancethat monoclonal antibodies will remain bound to the FVIII polypeptideand co-purify.

SUMMARY OF THE INVENTION

[0005] In the present invention, it has been found that diluting theeluate from the monoclonal antibody column provides certain advantagesin yield and/or reduced monoclonal antibody contamination of the FVIIIprotein being purified therefrom. Accordingly, the present inventionprovides improved methods for the purification of procoagulant proteins,including both FVIII and variants thereof, which may be produced byrecombinant techniques in higher yield and/or in more homogeneous form.

[0006] The present invention provides improved methods of purificationof FVIII protein from cell cultures, preferably from recombinant cellcultures. The methods provide for obtaining FVIII protein of a higherpurity than methods currently in use. In one embodiment, the methods ofthe present invention comprise diluting the eluate from theimmunoaffinity column with a solution of higher ionic strength than theeluate solution. In another embodiment, the methods of the presentinvention comprise diluting the eluate from an immunoaffniity columnwith a solution containing lower amounts of ethylene glycol thancontained in the eluate solution.

DETAILED DESCRIPTION OF THE INVENTION

[0007] Accordingly, the present invention provides improved methods forpurification of a Factor VIII polypeptide comprising:

[0008] a) adding a mixture containing Factor VM polypeptide to bepurified to an immunoaffinity matrix which binds by hydrophobicattraction to the FVIII polypeptide;

[0009] b) eluting the Factor VIII polypeptide from the immunoaffinitymatrix with a desorbing solution which causes desorption of the FactorVIII polypeptide, which is released in an elution solution;

[0010] c) diluting the elution solution with a solution comprisinghigher ionic strength than that of the elution solution, resulting in adiluted Factor VM solution;

[0011] d) passing the diluted Factor VIII solution through an ionexchange column capable of binding to the Factor VIII polypeptide,thereby binding the Factor VIII polypeptide while allowing contaminantsto pass through the ion exchange column; and

[0012] e) eluting the purified Factor VIII polypeptide from the ionexchange column.

[0013] The desorbing solution of step (b) may contain no salt, or verylow salt. The dilution of step (c) is preferably performed using asolution comprising from about 5 to about 20 mM NaCl, most preferablyabout 5 to about 15 mM NaCl. The eluting solution is preferably dilutedwith salt-containing solution from about 3-fold to about 5-fold, mostpreferably about 3-fold.

[0014] In another embodiment, the present invention comprises improvedmethods for purification of a Factor VIII polypeptide comprising:

[0015] a) adding a mixture containing Factor VIII polypeptide to bepurified to an immunoaffinity matrix which binds by hydrophobicattraction to the FVIII polypeptide;

[0016] b) eluting the Factor VIII polypeptide from the immunoaffinitymatrix with a desorbing solution which causes desorption of the FactorVIII polypeptide, which is released in an elution solution, wherein thedesorbing solution comprises a non-polar agent;

[0017] c) diluting the elution solution with a solution comprising lowerconcentration of the non-polar agent than that of the desorbingsolution, resulting in a diluted Factor VIII solution;

[0018] d) passing the diluted Factor VIII solution through an ionexchange column capable of binding to the Factor VIII polypeptide,thereby binding the Factor VIII polypeptide while allowing contaminantsto pass through the ion exchange column; and

[0019] e) eluting the purified Factor VIII polypeptide from the ionexchange column.

[0020] Preferably, the desorbing solution of step (b) contains ethyleneglycol, more preferably about 50% (v/v) ethylene glycol, and thedilution of step (c) is performed using a solution comprising less thanabout 50% (v/v) ethylene glycol, such that the final concentration ofethylene glycol is from about 17% to about 33% (v/v). In a preferredembodiment, the desorbing solution of step (b) contains 50% (v/v)ethylene glycol, and the dilution of step (c) is performed using asolution comprising no ethylene glycol, such that the finalconcentration of ethylene glycol is from about 17 to about 33% (v/v),most preferably about 33% (v/v) ethylene glycol. Preferably, the elutionsolution is diluted from about 1.5-fold to about 3-fold, most preferablyabout 1.5 fold, or 2:3.

[0021] The Factor VIII polypeptide of the present invention is generallyproduced recombinantly, but may also be purified from plasma. Therecombinant Factor VIII polypeptide may be natural full length FactorVIII polypeptide, or a variant, such as a B-domain deleted variant ofFactor VEII, including the VIII:SQ variant.

[0022] The immunoaffinity columns useful in the present invention may beany industrially acceptable column and resin, to which is adsorbed oneor more monoclonal or polyclonal antibodies which are capable of bindingto a Factor VIE polypeptide and in which the Factor VM polypeptide maylater be released using standard methods and reagents. Suitablemonoclonal antibodies, for example, are disclosed in Fass et al., Blood,59:594-600 (1982).

[0023] Useful as the desorbing substance is any non-polar agent, such asethylene glycol, dioxane, propylene glycol and polyethylene glycol, orany appropriate low ionic strength, low polarity buffered solution.

[0024] In preferred embodiments, the mixtures containing Factor VIIIpolypeptides may also include detergents and/or solvents, such aspolyoxyethyl detergents, including Triton X-100, Tween 80. In addition,the Factor VIII polypeptide containing solution may include bufferingsubstances, such as histidine.

EXAMPLE 1

[0025] Preparation of Recombinant Factor VIII:SQ

[0026] The production of recombinant factor VII:SQ (r-VII SQ) wasessentially performed as described in patent WO-A-9109122, [examples1-3]. A DHFR deficient CHO cell line (DG44NY) was electroporated with anexpression vector containing the r-VIII SQ gene and an expression vectorcontaining the dihydrofolate reductase gene. The conditioned medium(containing fetal calf serum) was clarified and then concentrated bytangential flow filtration. The solution was loaded onto an SP SepharoseFast Flow cation exchange resin, wherein the r-VIR SQ binds selectivelyto the resin through electrostatic forces.

[0027] The r-VIII SQ is eluted from the column at elevated ionicstrength by flowing elution solution (0.8 M NaCl, 3 mM EDTA, 0.02% (v/v)surfactant [Octoxynol 9], 0.1 MNH₄Ac, 5 mM CaCl₂, 1M Sorbitol, pH6.3+0.2) and is collected as a single UV adsorbing peak. The r-VIII SQis then put through a virus inactivation step employing thesolvent/detergent method using TNBP [Tri-(n-butyl)phosphate] andsurfactant [such as Octoxynol 9, Triton X-100).

[0028] The r-VIII SQ is next loaded onto an immunoaffinitychromatography gel, where the ligand was a monoclonal antibody (mAb,named 8A4) directed towards the heavy chain of factor VIII. Afterwashing, the factor VIII was eluted with a buffer containing 0.05 Mhistidine, 0.05 M CaCl₂ and 50% ethylene glycol and 0.02% Octoxynol 9(Tween), pH 6.6. The mAb eluate was loaded onto an anion exchangecolumn, Q Sepharose® FF sold by Pharmacia AB of Uppsala, Sweden. Afterwashing, the FVI SQ was eluted with a Q elution buffer containing 0.05 Mhistidine, 4 mM CaCl₂ 0.4 M NaCl, pH 6.3.

[0029] In order to improve upon the above purification system, severalseries of experiments were conducted to evaluate the effects on FVMrecovery of (a) dilution; (b) dilution with added NaCl; and (c) dilutionwith reduced, or with no, ethylene glycol.

[0030] Q Equilibration Buffer

[0031] The solution used to equilibrate the Q-column (the same as thedesorption buffer of the monoclonal antibody column) prior to loadingonto the ion exchange column comprises approximately the followingcomposition:

[0032] 0.05 M histidine

[0033] 0.05 M calcium chloride

[0034] 50% (v/v) ethylene glycol

[0035] 0.02% (v/v) Octoxynol 9 or other surfactant

[0036] pH 6.6+0.2

[0037] Series 1: Dilution with Q Equilibration Buffer

[0038] Following immunoaffinity purification, the eluate was dilutedfrom about 3-fold to about 5-fold with Q-equilibration buffer. In the3-fold dilution, total recovery of FVIII activity was acceptable, thoughreduced, while murine IgG detected in the eluate was very low. At higherdilutions, the loss of yield of FVII activity was significant.

[0039] Series 2: Dilution with 0 Equilibration Buffer Containing NaCl

[0040] Following immunoaffinity purification, the eluate was dilutedfrom about 3-fold to about 5-fold with Q-equilibration buffer containingNaCl in the range of about 7 to about 20 mM. Dilution generally producesa significant reduction in the amount of murine IgG recovered from theeluate prior to placing on the ion exchange column. Surprisingly, theaddition of NaCl also increased recovery of FVIII activity. Thisincrease in recovery was sufficient to offset the loss in recoveryresulting from dilution. The best results were observed in 3-fold to5-fold dilutions with NaCl in the range of about 10 to about 17 mM NaCl.The best recovery yields of FVIII activity were obtained with dilutionsof about 3-fold with about 15 mM NaCl. Dilution with less than about 7mM NaCl or greater than about 20 mM NaCl resulted in a loss of finalrecovery of FVIII activity.

[0041] The conclusion is that addition of about 7 mM to about 20 mM NaClto the Q Equilibration Buffer used to dilute the immunoaffinity eluaterestores the loss of yield associated with dilution without NaCl, whilealso producing beneficial results by reducing the murine antibodydetected in the eluate. In the most preferred embodiment, addition of QEquilibrium Buffer with about 15 mM NaCl added produced optimal results.

[0042] Series 3 and 4: Dilution with 0 Equilibration Buffer With No orReduced Ethylene Glycol

[0043] Following immunoaffinity purification, the eluate is diluted fromabout 1.5-fold to about 3-fold with Q Equilibration Buffer that does notcontain ethylene glycol, resulting in final ethylene glycol contentvarying from about 50% (v/v) in the Q Equilibration Buffer down to aslow as about 17% (v/v) in the 3-fold dilution without ethylene glycol. A1.5-fold dilution without ethylene glycol resulted in about a 33% (v/v)final ethylene glycol concentration. With decreased ethylene glycolconcentration, total recovery of protein increased over comparabledilution with Q Equilibration Buffer containing about 50% (v/v) ethyleneglycol.

EXAMPLE 2

[0044] 1.0 Introduction

[0045] A suitable downstream purification process for Factor VIII:SQ asproduced in Example 1 may consist of five chromatographic steps:cationic exchange (SP Sepharose FF), immunoaffinity (mAb Sepharose FF),anionic exchange (Q Sepharose FF), hydrophobic interaction (HIC, butylSepharose FF), and gel permeation chromatography (Superdex 200 pg). Theeluate from the mAb column may be directly loaded onto Q-Sepharose FFcolumn. A series of loading conditions on Q-Sepharose FF column wasexamined by PPD (in collaboration with P&U, Stockholm) to (I) study theimpact of the loading conditions on the activity recovery and thereduction in mouse IgG and HCP levels in the Q-Sepharose peak pool; (ii)establish optimal loading conditions on the anion exchanger. Results ofthis study are summarized in this Example.

[0046] 2.0 Experimental Procedures

[0047] 2.1 Materials:

[0048] Q-Sepharose FF resin was packed in a 79×5 mm ID Pharmacia HRcolumn. All buffers employed in this study were prepared by CTS byestablished procedures. The mAb peak pool from the purification processwere obtained frozen at −80° C. from P&U, Stockholm (LtE 923). The COBASassay kit and mega standard was bought from Chromogenix AB, Sweden.

[0049] 2.2. Procedures:

[0050] Q-Sepharose Scale Down Runs:

[0051] The Q-Sepharose FF column was initially equilibrated with 10 CVof buffer at a flow rate of 0.5 ml/min. Subsequently, the mAb peak poolwas diluted with the appropriate dilution buffer and loaded onto theQ-Sepharose FF column at a flow rate of 0.2 ml/mmin. The total activityunits loaded in all the experiments was 48,350 U/ml of the resin, and isclose to the upper limit specified in the PLA. The activity of the mAbpeak pool used to perform these experiments was 2860 IU/ml. The loadvolume in the 3-fold and 5-fold dilution experiments was therefore 78.6mls and 131 mls respectively. Following the load, the column was washedwith 40 CVs of a buffer containing 150 mM NaCl, 4 mM CaCl2, 50 mMHistidine, pH 6.6, at a flow rate of 0.32 ml/min (wash 2). The boundprotein was then eluted with a buffer containing 400 mM NaCl, 4 mMCaCl2, 50 mM Histidine, pH 6.3 at a flow rate of 0.05 ml/min. Wash 2 andelution in all the experiments were performed at a flow rate of 0.05ml/min. Wash 2 and elution in all the experiments were performed in thereverse direction. The column effluent during the various operations wascollected and assayed for activity. A 1.6 cv fraction was pooled duringelution beginning at the upward drift in the absorbance at 280 nm and istermed the peak pool. The load and peak pool samples were assayed formouse IgG and HCP levels by performing ELISA (P&U, Stockholm).

[0052] Regeneration:

[0053] The anion exchange column was regenerated, following each scaledown run, by passing five column volumes each of 2.0 M NaCl, 100 mMsodium phosphate (monobasic), pH 3.0 and 2.0M NaCl, 100 nM sodiumphosphate (dibasic), pH 11.0

[0054] Time Course Stability Studies:

[0055] The mAB peak pool was diluted different fold with (I) mAb elutionbuffer and (ii) mAb elution buffer containing 40 mM NaCl, and incubatedat room temperature. The activity in these samples was then assayed atdifferent time points.

[0056] 3.0 Results and Discussions

[0057] Time Course Stability Study:

[0058] The mAB peak pool was diluted 2-fold, 3-fold and 5-fold with mABelution buffer and incubated at room temperature. The drop in activityof these samples was monitored as a function of time. A modest drop of20% in activity was observed over the course of 24 hours. The loss inactivity was negligible at the end of 4 hours, and less than 10% at theend of 8 hours. Further, percentage drop in activity was observed to beindependent of the extent of dilution of the mAb peak pool and henceindependent of the solution concentration of FVIII in the mAb elutionbuffer. Similar results were obtained upon dilution of the mAb peak poolwith mAb elution buffer containing 40 mM NaCl.

[0059] Q-Sepharose Scale Down Experiments:

[0060] Results from the scale-down runs of the Q-Sepharose FF columnperformed with the mAb peak pool diluted 3-fold and 5-fold with the mAbelution buffer is shown in Table 1. TABLE 1 Dilution with mAb ElutionBuffer Load Activity at Flow Challenge Loading End of Run ThroughDilution (IU/ml Load Time (% of Initial Loss Fold resin) (IU/ml) (hours)Activity) (%) 3 48,350 953 6.55 82.8 3.6 5 48,350 572 10.9 70.4 3.3 WashWash Pre- Peak Post- Dilution #1 #2 Peak (1.6 cv) Peak Total Fold (%)(%) (%) (%) (%) Recovery 3 0.7 0.1 <0.1 57.4 3.9 65.8 5 0.4 0.1 <0.141.3 2.5 47.7

[0061] The flow-through losses in both cases was approximately 3.5% ofthe load, while the combined activity losses in the wash and prepeaksamples were less then 1%. The activity in the 1.6 cv peak pool for the3- and 5-fold dilution experiments were 57.5% and 41.3%, respectively,of the load, while the post-peak accounted for 3.9 and 2.5% of the loadactivity units, respectively. The corresponding values in manufacturingruns, wherein the mAb peak pool was loaded onto the column with nofurther modification of the eluate, is 5% in the flow-through and 70±9%in the peak pool. The other effluent streams have negligible activity.

[0062] These results clearly demonstrate that the yield across theQ-Sepharose FF column is sensitive to the extent of dilution of the mAbpeak pool prior to loading onto the column, and decreases withincreasing dilution. However, it is evident from the time coursestability studies that solution stability of FVIII:SQ is not affected bydilution. For a fixed number of activity units loaded onto the column,the operating time increases with dilution. As suggested by the timecourse stability data, a drop in yield can therefore be expected athigher load dilutions. Nevertheless, experimentally obtained activityvalues from the scale down runs was significantly lower than supportedby the time course stability data. One possible explanation is that theadsorption of FVIII:SQ onto the Q-Sepharose resin under diluteconditions leads to stronger interaction with the resin and has adenaturing effect on the protein, thereby leading to a lower recoveryupon elution. The yield at higher dilutions could then be improved byattenuating the ‘FVIII:SQ-resin’ interaction during loading. In order totest this hypothesis, subsequent experiments were performed with the mAbpeak pool diluted with mAb elution buffer containing NaCl.

[0063] Dilution with mAb Elution Buffer Containing NaCl:

[0064] The results from the Q-Sepharose scale down experiments performedusing mnAb peak pool diluted with mAb elution buffer containing variousconcentrations of NaCl is shown in Table 2. TABLE 2a 5-Fold DilutionWith mAb Elution Buffer Containing NaCl Load Activity Load NaClChallenge Loading at End of Run Flow Conc. (IU/ml Load Time (% ofInitial Through (mM) resin) (IU/ml) (hours) Activity) Loss (%) 10 48,350572 10.9 90.3 6.5 10 48,350 572 10.9 85.8 6.8 15 48,350 572 10.9 76.46.6 20 48,350 572 10.9 73.3 6.9 20 48,350 572 10.9 88.9 8.5 Load WashWash Pre- Peak Post- NaCl #1 #2 Peak (1.6 cv) Peak Total Conc. (%) (%)(%) (%) (%) Recovery 10 0.8 0.2 <0.1 73.1 1.8 82.4 10 0.9 0.2 <0.1 71.83.4 83.1 15 0.9 0.1 — 61.0 7.3 75.9 20 0.9 0.2 — 49.8 12.4 70.3 20 1.00.2 — 46.3 20.1 76.1

[0065] TABLE 2b 3-Fold Dilution With mAb Elution Buffer Containing NaClLoad Activity Load NaCl Challenge Loading at End of Run Flow Conc.(IU/ml Load Time (% of Initial Through (mM) resin) (IU/ml) (hours)Activity) Loss (%) 7 48,350 953 6.55 94.4 5.5 10 48,350 953 6.55 86.36.0 12.5 48,350 953 6.55 91.6 9.1 16.7 48,350 953 6.55 82.0 6.5 LoadWash Wash Pre- Peak Post- NaCl #1 #2 Peak (1.6 cv) Peak Total Conc. (%)(%) (%) (%) (%) Recovery 7 1.3 0.1 — 58.3 9.9 75.1 10 1.3 0.1 — 79.9 3.290.5 12.5 2.1 0.6 — 58.8 12.0 82.5 16.7 1.7 0.4 — 59.3 4.7 72.6

[0066] Loading the diluted mAb peak pool under conditions that attenuatethe ‘FVIII:SQ-resin’ interaction significantly increased the overallactivity recovery across the Q-Sepharose column. A greater fraction ofthis increase in activity was seen in the peak pool for the runsemploying 10 and 15 mM NaCl in the load, suggesting that there exists anoptimal NaCl concentration that leads to a maximum peak activityrecovery.

[0067] In the NaCl concentration range of about 7 to 20 mM, the activityloss in the flow through varied between 6.5 and 8.5%. These values aretwice of that seen in the 5-fold dilution run in the absence of NaCl.The combined wash and prepeak activity losses in all cases were lessthan 2%. The activity losses in the post-peak pool increases withincreasing NaCl concentration and was as high as 20% at an NaClconcentration of 20 mM. This is expected since the protein migratesfarther down the column during loading and subsequently takes longer toelute when the flow is reversed.

[0068] 3-Fold Dilution:

[0069] As in the case of 5-fold dilution, the overall activity recoveryand flow-through losses were higher when loaded in the presence of NaCl.The maximum overall and peak activity recovery was obtained at a NaClconcentration of 15 mM. However, existence of an optimum NaClconcentration is not as evident at this dilution level as it was at5-fold dilution.

[0070] Mouse IgG Results:

[0071] The mouse IgG data on the peak and post-peak pools for all 3- and5-fold dilution experiments are shown in Table 3: TABLE 3 Mouse IgG Datafrom 3-Fold and 5-Fold Dilution Experiments Dilution Load NaCl IgGLevels in Peak IgG Levels in Post- Fold Concentration Pool (ng/KIU) PeakPool (ng/KIU) 3-fold 0 0.8 2.1 7 0.5 2.0 10 0.8 5.3 12.5 0.7 1.8 16.71.2 3.6 5-fold 0 0.5 2.1 10 0.8 3.2 10 0.8 3.6 15 0.7 2.3 20 0.4 1.5 200.6 1.3

[0072] The IgG values in the peak pool for the 3-fold dilution runsvaried from 0.5 to 1.2 ng/KIU and 0.5 to 0.8 ng/KIU for the 5-folddilution runs. The corresponding values in manufacturing runs, whereinthe mAb peak pool was loaded onto the column with no furthermodification of the eluate, averaged 2.3 ng/KIU. Thus, dilution of themAb peak pool with mAb elution buffer, with or without NaCl, prior toloading reduced the IgG levels in the Q-Sepharose peak pool. Thiseffect, beyond the mere dilution of IgG levels, may be the result of agiven association constant for formation of IgG-FVIII:SQ complex. Thus,lowering the concentrations of IgG and FVIII:SQ lowers the concentrationof the complex, thereby allowing greater removal of IgG across the ionexchanger. In both the 3-fold and 5-fold dilution experiments, nocorrelation was observed between IgG values in the Q-Sepharose peak pooland NaCl concentrations in the load. Thus, in the range of NaClconcentrations employed in these experiments, addition of NaCl does notappear to provide additional reduction in mouse IgG levels.

[0073] Host Cell Protein Results:

[0074] The host cell protein data on the peak pool for the 3-fold and5-fold dilution experiments are shown in Table 4: TABLE 4 Host CellProtein (HCP) Levels in 3-Fold and 5-Fold Dilution Experiments DilutionFold Load NaCl Conc (mM) HCP in Peak Pool (ng/KIU) 3 7 10.3 12.5 4.2 510 14.1 15 9.9 20 10.9

[0075] The corresponding values in manufacturing runs, wherein the mAbpeak pool was loaded onto the column with no further modification of theeluate, averaged 20 ng/KIU. These results suggest that the HCP levels inthe peak pool decrease with increasing NaCl concentrations, and areindependent of the extent of dilution. The addition of NaCl mayattenuate the binding of HCP to the resin, and therefore allow lowerlevels of HCP in the Q-Sepharose peak pool.

[0076] 4.0 Conclusions

[0077] Dilution of the mAb peak pool with mAb elution buffer prior toloading on Q-Sepharose column significantly decreased the yield acrossthis step. The decrease in yield is an increasing function of the extentof dilution. However, the solution stability of FVIII is independent ofthe extent of dilution with mAb elution buffer, thereby suggesting thatloading under dilute conditions leads to a stronger ‘FVM-resin’interaction and has a denaturing effect on the protein. Addition ofsodium chloride to the dilution buffer attenuates the ‘FVIII-resin’interaction and increases the yield across the Q-Sepharose column.Increasing the NaCl concentrations, however, increases the flow-throughand post-peak losses and hence there exists an optimum NaClconcentration at which the yield losses are siguificantly offset. Theoptimum concentration for the 3-fold and 5-fold dilution runs appears tobe in the 7 to 20 mM concentration, more particularly about 15 mM.

[0078] Diluting the mAb peak pool with mAb elution buffer also reducedthe IgG and HCP levels in the Q-Sepharose peak pool. In theconcentration range of NaCl examined, HCP levels in the Q-Sepharose peakpool decreased with increasing NaCl concentrations in the load. Overall,a combination of dilution of the mAb peak pool and adding NaCl atconcentrations of 7 to 20 mM resulted in improved purification withoutsignificant yield loss.

What is claimed is:
 1. An improved method for purification of a FactorVIII polypeptide comprising: a) adding a mixture containing Factor VIIIpolypeptide to be purified to an immunoaffinity matrix which binds byhydrophobic attraction to the FVII polypeptide; b) eluting the FactorVIU polypeptide from the immunoaffinity matrix with a desorbing solutionwhich causes desorption of the Factor VII polypeptide, which is releasedin an elution solution; c) diluting the desorbing solution with asolution comprising higher ionic strength than that of the elutionsolution, resulting in a diluted Factor VIII solution; d) passing thediluted Factor VIII solution through an ion exchange column capable ofbinding to the Factor VII polypeptide, thereby binding the Factor VIIIpolypeptide while allowing contaminants to pass through the ion exchangecolumn; and e) eluting the purified Factor VM polypeptide from the ionexchange column.
 2. A method of claim 1, wherein the desorbing solutionof step (b) contains no salt, and the dilution of step (c) is performedusing a solution comprising from about 7 to about 20 mM NaCl.
 3. Amethod of claim 1, wherein the desorbing solution of step (b) containsno salt, and the dilution of step (c) is performed using a solutioncomprising about 15 mM NaCl.
 4. A method of claim 3, wherein thedesorbing solution is diluted from about 3-fold to about 5-fold.
 5. Amethod of claim 3, wherein the desorbing solution is diluted about3-fold.
 6. An improved method for purification of a Factor VIIIpolypeptide comprising: a) adding a mixture containing Factor VIIIpolypeptide to be purified to an immunoaffinity matrix which binds byhydrophobic attraction to the FVIII polypeptide; b) eluting the FactorVIII polypeptide from the immunoaffinity matrix with a desorbingsolution which causes desorption of the Factor VIm polypeptide, which isreleased in an elution solution, wherein the desorbing solutioncomprises a non-polar agent; c) diluting the elution solution with asolution comprising lower concentration of the non-polar agent than thatof the elution solution, resulting in a diluted Factor VIII solution; d)passing the diluted Factor VIII solution through an ion exchange columncapable of binding to the Factor VIII polypeptide, thereby binding theFactor VIII polypeptide while allowing contaminants to pass through theion exchange column; and e) eluting the purified Factor VIII polypeptidefrom the ion exchange column.
 7. A method of claim 6, wherein thedesorbing solution of step (b) contains 50% (v/v) ethylene glycol, andthe dilution of step (c) is performed using a solution comprising lessthan 50% (v/v) ethylene glycol, such that the final concentration ofethylene glycol is from about 17% to about 33% (v/v).
 8. A method ofclaim 6, wherein the desorbing solution of step (b) contains 50% (v/v)ethylene glycol, and the dilution of step (c) is performed using asolution comprising no ethylene glycol, such that the finalconcentration of ethylene glycol is from about 17 to about 33% (v/v). 9.A method of claim 8, wherein the desorbing solution is diluted fromabout 1.5-fold to about 3-fold.